This invention relates to a method of modifying a glycoprotein's oligosaccharide structure.
Many proteins of biological and pharmaceutical interest have oligosaccharides attached to their polypeptide backbone. Although proteins produced by E. coli and other bacteria are non-glycosylated, proteins secreted by yeasts and mammalian cells are normally glycosylated. The sugar chains of these glycoproteins can be attached by an N-glycosidic bond to the amide group of asparagine residues (Asn-linked oligosaccharides) or by an O-glycosidic bond to the hydroxyl group of serine or threonine residues (Ser- or Thr-linked oligosaccharides).
In the Asn-linked oligosaccharides, the types of structures found on native proteins can be generally classified as high mannose, hybrid and complex type sugar chains. However, considerable variation in these basic structures is common. See, for example, the 16 oligosaccharide structures on a tissue plasminogen activator derived from normal human colon fibroblast cells as described in U.S. Pat. No. 4,751,084. Further background information on the assembly of Asn-linked oligosaccharides can be had by reference to Kornfeld and Kornfeld, Ann. Rev. Biochem. 54, 631-664 (1985).
The carbohydrate structure of a glycoprotein can have a significant effect upon its biological activity. That is, the oligosaccharides can affect the protein's antigenicity, stability, solubility and tertiary structure. The carbohydrate side-chains also can influence the protein's half-life and target it to receptors on the appropriate cells. The carbohydrate residues can affect both inter- and intracellular recognition. The sugar groups thus can control the relative effectiveness of a therapeutic protein when administered to a patient. These and other such functions of the carbohydrate moiety of glycoproteins are discussed, for example, by Delente, Trends in Biotech. 3(9), 218 (1985); van Brunt, Bio/Technology 4, 835-839 (1986); and Taunton-Rigby, Biotech USA 1988, Proc. Conf. San Francisco, Nov. 14-16, 1988, pp. 168-176.
It is also apparent that differences in the glycosylation pattern (i.e., particular structure at a specific site) on similar proteins or proteins with identical amino acid sequences can have profound effects on antigenicity, metabolism and other physiological properties. See, for example, the association of rheumatoid arthritis and osteoarthritis with changes in the glycosylation pattern of total serum by Parekh et al., Nature 316, 452-457 (1985) and in U.S. Pat. No. 4,659,659.
Another example of a glycoprotein in which significant biological activity resides in the oligosaccharide moieties is that of human chorionic gonadotropan (hCG). Thus, it is known that hCG without carbohydrate is a competitive inhibitor of native hCG; that oligosaccharides isolated from hCG inhibit action of native hCG; and that tumor-produced hCG having the same amino acid sequence as native hCG but different sugars has almost no biological activity. See Calvo et al., Biochemistry 24, 1953-1959 (1985); Chen et al., J. Biol. Chem. 257, 14446-14452 (1982).
Yet another group of proteins in which the presence and/or structure of the oligosaccharides can have important biological effects are the plasminogen activators (PA), namely urokinase (u-PA) and tissue plasminogen activator (t-PA). The functional properties of carbohydrate-depleted t-PA are discussed ty Little et al., Biochemistry 23, 6191-6195 (1984), and by Opdenakker et al., "EMBO Workshop on Plasminogen Activators," Amalfi, Italy, Oct. 14-18, 1985. The latter scientists report that enzymatic cleavage of carbohydrate side-chains from melanoma (Bowes) derived t-PA by treatment with .alpha.-mannosidase causes an increase in the biologic activity of the modified t-PA. The Bowes melanoma t-PA is a glycoprotein which has a molecular weight of about 68,000-70,000 daltons and a 527 amino acid structure with serine at the NH.sub.2 -terminus. The melanoma t-PA can exist as two chains, an A-chain and a B-chain. It also separates into two variants (or isoforms) in the A-chain, known as types I and II, which differ by about M.sub.r 2000-3000. See Ranby et al., FEBS Lett. 146 (2), 289-292 (1982), and Wallen et al., Eur. J. Biochem. 132, 681-686 (1983). Type I is glycosylated at Asn-117, Asn-184 and Asn-448, whereas Type II is glycosylated only at Asn-117 and Asn-448 according to Pohl et al., Biochemistry 23, 3701-3703 (1984). A high mannose structure has been assigned to Asn-117, whereas two complex carbohydrate structures are assigned to Asn-184 and Asn-448 by Pohl et al., "EMBO Workshop on Plasminogen Activators," Amalfi, Italy, Oct. 14-18, 1985, and Eur. J. Biochem. 170, 69-75 (1987).
It is known that the normal t-PA molecule has five functional domains or regions: A fibronectin-like finger domain (F); an epidermal growth factor region (GF); two kringle regions (K1 and K2); and a serine protease region (SP). The full t-PA molecule thus can be represented as F+GF+K1+K2+SP. In the 527 amino acid sequence of the normal t-PA molecule described by Pennica et al., Nature 301, 214-221 (1983), the finger region comprises residues 1-43; the growth factor region comprises residues 44-91; kringle refers to a characteristic triple disulfide structure of which t-PA has two such regions, K1 - residues 92-173, and K2 - residues 180-261; and the serine protease comprises residues 262-527. The SP catalytic site is formed from the His-322, Asp-371 and Ser-478 residues. Various deletions of one or more of these regions together with elimination of one or more of the glycosylation sites such as by site-directed mutagenesis have been described heretofore.
In European Patent Application 178,105, published Apr. 16, 1986, a modified t-PA is described in which one or more of the glycosylation sites have been eliminated by site-directed mutagenesis of Asn to Gln at the glycosylation sites in the kringle and serine protease regions. The amino acid residues Asn-120, -187 and -451 in the described uterine t-PA are equivalent to residues Asn-117, -184 and -448, respectively, in the Bowes melanoma t-PA.
A variety of site-mutagens are also described in European Patent Application 227,462, published July 1, 1987, including mutagenesis at the above glycosylation sites and at the cleavage sites in the region 272-280, especially in the sequence Phe(274)-Arg(275)-Ile(276)-Lys(277).
According to European Patent Application 238,304, published Sept. 23, 1987, melanoma t-PA devoid of carbohydrate structure at amino acid residue 117 but unmodified from native t-PA in functional carbohydrate structure at amino acid residues 184 and/or 448 retains substantially full biological activity compared to native t-PA but has increased in vivo half-life. See also Hotchkiss et al., Thromb. Haemostasis 60, 255-261 (1988).
In U.S. Pat. No. 4,751,084, a glycosylated t-PA obtained from cultured normal human colon fibroblast cells was found to have a unique, heterogeneous glycosylation pattern that differs significantly from the t-PA of Bowes melanoma although the protein moieties are substantially similar. Differences in biological activities such as thermal stability and fibrin stimulatory properties were shown to be caused by the specific glycoforms present.
The role of specific sugar units on the clearance of t-PA from circulation is further discussed, for example, by Lucore et al., Circulation 77 (4), 906-914 (1988).
It is thus apparent that methods of modifying a glycoprotein's oligosaccharide structure can have substantial importance to protein research for the development of biopharmaceuticals through carbohydrate engineering.